JP6382488B2 - Solid phase peptide synthesis via side chain bonds - Google Patents

Description

  The present invention relates to peptide synthesis.

High purity peptides and peptaibols by solid phase peptide synthesis using hydroxy amino acids, hydroxy amino acid amides, hydroxy amino alcohols or small peptides containing hydroxy amino acids attached to the summary polymer via side chains as starting resins was gotten.

Definitions and abbreviations “Hya” or “hydroxyl amino acid” mean an amino acid containing a hydroxyl (—OH) group.
The N-terminus or amino terminus is the first amino acid in the peptide chain.
The C-terminus or carboxy terminus is the last amino acid in the peptide chain and is shown below.

  “P” or “solid support” or “resin” means an insoluble material containing functional groups suitable for reacting or linking with amino acids or peptides. Such solid supports or resins are well known in the art.

“Alkyl”, for example C _1-10 -alkyl or C _1-6 -alkyl, is a branched or unbranched, fully saturated acyclic hydrocarbon group (ie containing double or triple bonds). Not composed of carbon and hydrogen). In some embodiments, the alkyl may be substituted or unsubstituted. Alkyl includes, but is not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like, and in some embodiments each of them is optionally May be substituted. Alkyl substituents include, but are not limited to, C _1-3 -alkoxy, halogen (F, Cl, Br or I), nitro, amino, —SH and —OH.

“Attachment” means the linking of an amino acid or peptide or peptide derivative to an insoluble support.
“Hse” means homoserine and “Hnv” means hydroxyl norvaline.
“SPPS” or “solid phase peptide synthesis” means the synthesis of peptides with the use of a resin as described herein.
“PNA” means 4-nitroanilide.
“DME” means dimethoxyethane.

“Acid sensitive resin” means an insoluble material or resin containing functional groups suitable for reaction or linkage with an amino acid or peptide, which can be cleaved from the peptide by acidic treatment.
“Acid sensitive protecting group” means a protecting group that can be cleaved from an amino acid or peptide or peptide derivative by acidic treatment or under acidic conditions.
“Peptaibol” means a peptide that contains an amino alcohol rather than an amino acid or amino acid amide at its C-terminal position.

“Step-by-step” means a peptide synthesis method in which each of the amino acids contained in the peptide chain is individually and sequentially introduced. This method may or may not include an intermediate purification step.
“Protected peptide” means a peptide in which all functional groups are protected or blocked by protecting groups.
“Partially protected peptide” means a peptide in which at least one functional group is protected or blocked by a protecting group.

  Solid phase peptide synthesis traditionally involves the attachment of a C-terminal amino acid to its appropriate solid support via its α-carboxyl functional group, and the sequential addition of amino acid residues in a growing peptide chain. By extending the peptide chain in the direction of the amino terminus. Hundreds of thousands of publications and patents describe this method and its application to the production of peptide drugs.

  Contrary to the attachment of the C-terminal carboxyl functionality, attachment of amino acids and peptides to the appropriate resin via the amino acid side chain and its application in SPPS is very short, especially in less than 30 publications and patents. Have been described. Most of these publications describe the attachment of amino acids via the side chain carboxyl functionality of Asp and Glu.

  The inventor's knowledge has limited side chain attachment of amino acids via the side chain hydroxyl function and application in peptide synthesis: side chain attachment of Fmoc-Hya-pNA (formula A1) [A. Bernhardt, M. Drewello and M. Schutkowski, The solid-phase synthesis of side-chain-phosphorylated peptide-4-nitroanilides J. Peptide Res. 50, 1997. 143-152] and their use for the synthesis of short nitroanilide substrates Synthesis of Fmoc-Hya-O-allyl ester on 2-chlorotrityl resin with the aid of microwave for application in cyclic peptide preparation (formula A2 [L. Rizzi, K. Cendic, N. Vaiana, S. Romeo, Alcohols immobilization onto 2-chlorotritylchloride resin under microwave irradiation, Tetrahedron Letters 52 (2011) 2808-2811]) -O-methyl ester synthesis ( Formula A3 [C. Cabrele, M. Langer and AG Beck-Sickinger, Amino Acid Side Chain Attachment Approach and Its Application to the Synthesis of Tyrosine-Containing Cyclic Peptides, J. Org. Chem. 1999, 64, 4353-4361]) And their application for the synthesis of short cyclic peptides.

  The inventor's knowledge does not disclose Hse and Hyp side chain attachment. Furthermore, the application of Hya side chain mounted on acid sensitive resins for solid phase synthesis of protected peptides, protected peptide fragments and protected peptide amides and peptaibols has not been reported.

Solid Phase Peptide Synthesis In one embodiment, an improved synthesis of pharmaceutically interesting peptide acids, peptide amides, and peptaibols is provided.

In one aspect of the present invention, amino acid-resin conjugates of formulas I-IV with a hydroxy amino acid attached via its amino acid side chain or a small peptide containing a hydroxy amino acid in sequence on a trityl or benzhydryl type resin Alternatively, peptides were produced very effectively in high yield and purity by generating peptide resin conjugates. Here, in Formulas I-IV, P is selected from the support used in solid phase peptide synthesis and Pr ¹ is an amino protecting group selected from H or Fmoc, Boc, Trt, Dde and Alloc. , Pr ² is an acid sensitive hydroxy protecting group selected from Trt, Clt, Mmt, Mtt, Dpm and tBu, and Hya is selected from D- or L-Ser, Thr, Tyr, Hse, Hyp, Hnv, etc. And A is an acid-sensitive alkoxy group selected from OH, OTrt, OClt, OMmt, OMtt, ODpm and OtBu, NH ₂ , NHR ¹ , NR ¹ R ² (where R ¹ and R ² is independently an alkyl group or a protected or semi-protected peptide containing 1 to 10 amino acids in the sequence.

In other embodiments, the inventor has Formulas III-VI selected from amino alcohols derived from natural hydroxy amino acids, wherein P, X, V, Z and Pr ¹ are as defined above; R ³ and R ⁴ are alkyl, aryl or aralkyl groups, and Pr ² is a trityl, benzhydryl or benzyl type acid sensitive protecting group) by solid phase synthesis using a resin bound amino alcohol, such as peptraibol eg octreotide Disclose that (octreotide) was obtained.

  Furthermore, the present inventors have found that when a peptide prepared by application of a resin of formulas I-IV is attached on a trityl-type resin via the side chain hydroxyl function of a hydroxy amino acid, the resin is subjected to mild acid treatment. It is first disclosed that tBu and benzyl-type side chain protecting groups remain intact. In one aspect, cleavage from the resin occurs by treatment with a 1-3% acid solution such as TFA, dilute HCl solution, optionally with the addition of a scavenger in a solvent. In one aspect, the cleavage can be performed in a solvent such as DCM or acetone. Such partially protected peptides have been found useful in the synthesis of longer peptides by fragment condensation in solution or on the solid phase. The method of the present invention extends the versatility of application of the resins described herein, and also provides a significant improvement in the purity of the resulting pharmaceutical peptides and at the same time substantially reduces the cost of their synthesis. Let

  Some peptides of pharmaceutical interest represent step-by-step methods or fragment condensation in solution and on the solid phase, or combinations thereof, as representative of the novel methods described herein Manufactured by. The following examples are representative examples, and application to other peptides is not limited at all.

Lanreotide :
In one embodiment, lanreotide was produced by solid phase synthesis using a resin-bound Thr-amide as shown below.

Human insulin B chain :
Optionally, human insulin B chain was synthesized by SPPS. In one aspect, the synthesis begins with Thr-t-butyl coupled to a resin as described in the Examples using 4-methoxybenzhydryl resin. Optionally, the synthesis also includes 1-8 partially protected Boc-Phe-Val-Asn (Trt) -Gln (Trt) -His (Trt) -Leu-Cys (Trt) -Gly-OH on the solid phase. The fragment can be performed by condensation with a 9-30 fragment attached to the resin, or after cleavage of the partially protected 9-30 fragment from the resin, in a solution of 1-8 and 9-30 fragments Can be carried out by condensation.

Salmon calcitonin :
Optionally, salmon calcitonin can be produced by initiating synthesis from Fmoc-Thr-Pro-NH ₂ bound to a resin. The peptide chain is then extended using Fmoc-amino acids.

  Optionally, salmon calcitonin bound to the resin is prepared by fragment condensation on the resin as shown above or in solution as shown below, using 2-4 fragments.

Octreotide :
In another embodiment, octreotide is attached to Fmoc-threoninol-OTrt to 4-methoxybenzhydryl resin via the side chain of threoninol as shown below, followed by Fmoc-amino acid It was efficiently synthesized by assembly of octreotide chains and finally cleavage of octreotide from the resin using sequential or simultaneous Cys-oxidation. Fmoc-threoninol-OTrt is much easier to manufacture than Fmoc-Thr (tBu) -ol, which can be mounted on the resin via the hydroxymethyl group of threoninol on a suitable resin. This is because H-Thr (OtBu) -ol, which is used as a starting material for the production of Fmoc-Thr (tBu) -ol, is used to attach threoninol via a side chain onto the resin. This is because it is very difficult to produce compared to Fmoc-threoninol-OTrt.

Exenatide :
In other examples, Fmoc-Ser-NH ₂ is attached to the trityl resin via its side chain and used for the synthesis of exenatide. This synthesis is performed in a step-by-step manner, either in solution or on solid phase after cleavage of the partially protected exenatide fragment from the resin by mild acid treatment, as described below. Or by fragment condensation. According to this method, most impurities typically produced during the synthesis of peptides containing many Pro and Gly residues are completely avoided and high purity peptides are obtained. This method also allows complete avoidance of impurities resulting from cleavage of the peptide from the peptide amide linker using other methods known in the art. The above-described methods known in the art significantly reduce peptide yield and purity.

  In one aspect, exenatide cleaves partially protected peptide 12-39 from the resin and condenses it with the partially protected 1-11 fragment in solution as shown below. Can be manufactured. Alternatively, condensation can be performed using fragment 1-13 and fragment 14-39 to give the protected exenatide.

Pramlintide :
This method is also very effectively used in the production of amylin peptides. In one aspect, side chain attachment can be accomplished using one of the C-terminal Ser, Thr or Tyr residues of amylin or its derivatives, such as pramlintide. The synthesis can be carried out in a step-by-step manner in solution or on the solid phase or by fragment condensation. By introducing pseudoprolines (see Ψ, Mutter et al, Peptide Res. (1995 8, 145)) into the growing peptide chain, synthesis is accelerated and the purity of the resulting peptide is increased. Improved.

Alternatively, the synthesis of pramlintide can be performed in the liquid phase with similar success with respect to the purity and yield of pramlintide. In one embodiment, the protected peptide bound by the resin via the side chain of Fmoc-Tyr-NH ₂ is used to leave the tBu-type side chain protecting group intact and use mild acid treatment to remove the peptide chain. It can be cleaved quantitatively from the resin at various locations. In one example, as shown below, a partially protected 1-10 fragment prepared on 2-chlorotrityl resin is converted to a partially protected 11-37 fragment amide in a stepwise (step Successfully condensed in a (by-step) manner.

Pramlintide :

Tetracosactide (ACTH 1-24) :
In other examples, as shown below, ACTH 1-24 is protected by a stepwise (step-by-step) method or partially starting from resin-bound Fmoc-Tyr-Pro-OtBu. 1-10 fragments were effectively prepared by condensing in solution with 11-24 fragments or resin-bound 11-24 fragments.

Bivalirudin :
In other examples, as shown below, bivalirudin extends the peptide chain in a step-by-step manner with Fmoc-amino acids starting from resin-bound Fmoc-Tyr-Leu-OtBu. Finally, it was produced in high yield and purity by deprotecting and cleaving the peptide from the resin.

  Alternatively, bivalirudin can be obtained by condensing a protected fragment on the resin, or by cleaving a partially protected peptide containing 4-15 amino acid residues from the resin and making it contain 5-16 amino acids. It was obtained by condensing with rubine fragments in solution. Synthesis of bivalirudin by fragment condensation on a resin between a partially protected 1-10 bivalirudin fragment and a partially protected 11-20 bivalirudin fragment attached to the resin is described below. .

Example 1. Preparation of Fmoc-Thr (4-methoxybenzhydrylpolystyryl) -OtBu

  30 mmol of Fmoc-Thr-OtBu prepared by a conventional reaction between H-Thr-OtBu and Fmoc-OSu, and 20 g (30 mmol) of 4-methoxybenzhydrylpolystyrene resin (CBL-Patras product) And 60 mmol of DIPEA in 250 ml of THF for 10 hours at room temperature. Next, 60 mmol of methanol was added to the mixture and the mixture was shaken for an additional 4 hours. The resin was filtered and washed 3 times with THF / MeOH / DIPEA (85: 10: 5), 6 times with DMF, 4 times with IPA, 3 times with DEE and vacuum dried to constant weight. 29 g of resin-bound Fmoc-Thr-OtBu was obtained with a loading of 0.95 mmol / g resin.

Example 2. Fmoc-Thr (4-Methoxybenzhydrylpolystyryl) -O-Clt

30 mmol of Trt-Thr-OMe prepared by a conventional reaction of H-Thr-OMe with Trt-Cl / Me ₃ SiCl and DIPEA was converted into 20 g (30 mmol) of 4-methoxy 4 ′ polystyrylbenzhi The reaction was performed with drill bromide resin (CBL-Patras product) and 60 mmol DIPEA in 250 ml THF for 10 hours at room temperature. Next, 60 mmol of methanol was added to the mixture and the mixture was shaken for an additional 4 hours. The resin was filtered and three times with THF / MeOH / DIPEA (85: 10: 5), three times with DCM, three times with 1% TFA in DCM, four times with THF, THF / water / methanol (70:15 : 15) 3 times with 1N-LiOH, 3 times with THF / water (75:25), 4 times with DMF and then 2 hours at room temperature for 2 hours, 60 mmol Fmoc-OSu and 30 mmol DIPEA And then washed 3 times with DMF, 3 times with DCM, and then reacted with 50 mmol Trt-Cl and 50 mmol DIPEA for 3 hours at room temperature, washed 4 times with DMF and 6 times with DEE. And vacuum dried to constant weight. With a load of 0.78 mmol / g resin, 32.3 g of resin bound Fmoc-Thr-OtBu was obtained.

Example 3. Fmoc-Throl (4-methoxybenzhydrylpolystyryl) -O-Clt
A) Depart from Fmoc-threoninol

  50 mmol of commercial Fmoc-threoninol (CBL-Patras) in 350 ml DCM was reacted with 55 mmol monomer Clt-Cl and 55 mmol DIPEA at room temperature for 4 hours. The resulting mixture was extracted with water as usual and the DCM phase was dried over anhydrous sodium sulfate and filtered. To the resulting solution was added 30 g 4-methoxy, 4-polystyryl benzhydryl bromide (CBL-Patras) and 50 mmol DIPEA and the resulting mixture was stirred at room temperature for 4 hours. The resin was filtered and washed 6 times with DMF, 4 times with IPA and 4 times with DEE, and vacuum dried to constant weight. With a load of 0.82 mmol / g, 38.4 g of resin-bound Fmoc-threoninol was obtained.

B) Departure from Trt-Thr (Resin) -OMe

30 mmol of Trt-Thr-OMe prepared by the reaction of H-Thr-OMe with Trt-Cl / Me ₃ SiCl and DIPEA according to a conventional method was added to 20 g (30 mmol of 4-methoxy4′-polystyrylbenz Reaction with hydryl bromide resin (CBL-Patras product) and 60 mmol DIPEA in 250 ml THF for 10 hours at room temperature, then 60 mmol methanol was added to the mixture and the mixture The resin was filtered and washed 3 times with THF / MeOH / DIPEA (85: 10: 5), 5 times with THF and reacted with 30 mmol LiBH _{4 in} THF.

  The resin is then filtered and washed 6 times with THF, 4 times with DCM, 6 times with 1% TFA in DCM, 3 times with DMF / DIPEA (97: 3) and then 2 hours at room temperature. , Reacted with 60 mmol Fmoc-OSu and 30 mmol DIPEA, washed 3 times with DMF, 3 times with DCM and then reacted with 50 mmol Clt-Cl and 50 mmol DIPEA for 3 hours at room temperature. Washed 4 times with DMF, 6 times with IPA and 6 times with DEE and vacuum dried to constant weight. With a loading of 0.74 mmol / g resin, 34.7 g of resin-bound Fmoc-Throl-O-Clt was obtained.

Example 4. Fmoc-Ser (trityl resin) -NH ₂
50 mmol Fmoc-Ser-NH ₂ prepared by standard procedures known in the art was dissolved in 0.5 liter DCM. To this suspension, 30 g of trityl chloride resin (36 mmol) and 65 mmol of DIPEA were added and the mixture was stirred at room temperature for 6 hours. In succession 25 ml of methanol and 30 mmol of DIPEA were added and the mixture was further stirred at room temperature for 2 hours. The resin was then filtered and washed 3 times with DCM / MeOH / DIPEA (90: 5: 5), 5 times with DMF, 4 times with IPA, 4 times with DEE and vacuum dried to constant weight. 41.1 g Fmoc-Ser-NH ₂ -containing resin was obtained with a load of 0.71 mmol / g.

Example 5. Fmoc-Tyr (2-chlorotrityl resin) -NH ₂
According to the above procedure, 43.7 g of resin was obtained from 50 mmol Fmoc-Tyr-NH ₂ and 30 g 2-CTC chloride resin with a loading of 0.81 g Tyr / g resin.

Example 6. Fmoc-Hyp (4-methylbenzhydryl resin) -NH ₂
Following the above procedure, 39.8 g of resin was obtained from 50 mmol Fmoc-Hyp-NH ₂ and 30 g 4-methylbenzhydryl bromide resin with a loading of 0.49 g Hyp / g resin.

Example 7. Fmoc-Thr (4-methoxybenzhydryl resin) -Pro-NH ₂
50 mmol Fmoc-Thr-Pro-NH ₂ prepared by coupling Fmoc-Thr (tBu) -OH and H-Pro-NH ₂ according to standard procedures known in the art, 0.5 liter DME Dissolved in. To the resulting solution, 30 g of 4-methoxybenzhydryl bromide resin (45 mmol) and 65 mmol of DIPEA were added and the mixture was stirred at room temperature for 6 hours. Then 25 ml methanol and 50 mmol DIPEA were added and the mixture was stirred at room temperature for 2 hours. The resin was then filtered and washed 3 times with DME / MeOH / DIPEA (90: 5: 5), 5 times with DMF, 4 times with IPA, 4 times with DEE and vacuum dried to constant weight. With a load of 0.77 mmol / g, 44.5 g of Fmoc-Thr-Pro-NH ₂ containing resin was obtained.

Example 8. Fmoc-Tyr (2-chlorotrityl resin) -Pro-OtBu
50 mmol Fmoc-Tyr-Pro-OtBu was prepared and dissolved in 0.5 liter DCM according to standard procedures known in the art. To the resulting solution, 30 g of 2-chlorotrityl chloride resin (48 mmol) and 65 mmol of DIPEA were added and the mixture was stirred at room temperature for 12 hours. Then 25 ml of methanol and 50 mmol of DIPEA were added and the mixture was stirred for another 2 hours at room temperature. The resin was then filtered and washed 3 times with DCM / MeOH / DIPEA (90: 5: 5), 5 times with DMF, 4 times with IPA, 4 times with DEE and vacuum dried to constant weight. 44.5 g of Fmoc-Tyr-Pro-OtBu-containing resin was obtained with a load of 0.64 mmol / g.

Example 9. Fmoc-Tyr (2-chlorotrityl resin) -Leu-OtBu
50 mmol Fmoc-Tyr-Leu-OtBu prepared according to standard procedures known in the art was dissolved in 0.5 liter THF. To the resulting solution, 30 g of 2-CTC chloride resin (48 mmol) and 65 mmol of DIPEA were added and the mixture was stirred at 60 ° C. for 12 hours. Then 25 ml of methanol and 50 mmol of DIPEA were added and the mixture was stirred at room temperature for a further 2 hours. The resin was then filtered and washed 3 times with DCM / MeOH / DIPEA (90: 5: 5), 5 times with DMF, 4 times with IPA, 4 times with DEE and vacuum dried to constant weight. With a load of 0.64 mmol / g, 44.5 g of Fmoc-Tyr-Leu-OtBu-containing resin was obtained.

Example 10 Solid Phase Synthesis of Peptides and Protected Peptide Segments
General procedure
A1. Preparation of Loaded 2-Chlorotrityl Resin General Procedure 2-Chlorotrityl chloride resin (CTC-Cl) (100 g; load 1.6 mmol / g) (CBL-Patras) into a 2 L peptide synthesis reactor And swell with 700 mL of dichloromethane (DCM): dimethylformamide (DMF) 1: 1 at 25 ° C. for 30 minutes. The resin is filtered and a solution of 100 mmol Fmoc-amino acid and 300 mmol diisopropylethylamine (DIEA) in 500 mL DCM is added. The mixture is stirred at 25 ° C. for 2 hours under nitrogen.

  The remaining active sites of the 2-CTC resin are then neutralized by addition of 10 mL of methanol (MeOH) and reaction for 1 hour. The resin is filtered and washed twice with 400 mL DMF. The resin is filtered and washed twice with 500 mL of 25% by volume piperidine in DMF for 30 minutes. The resin is then treated 4 times with 500 mL DMF. The resin is de-swelled by three washes with 500 mL isopropanol (IPA). Dry the resin to constant weight. To this resin, 70-95% of the mmol of amino acid used was bound.

B. Solid Phase Synthesis As described in General Protocol Part A or Example 1, using 1.0 g amino acid or peptide esterified or attached via a side chain to a trityl or benzhydryl type resin Solid phase synthesis was performed at The following protocol was used in the synthesis.

B1. Resin swelling The resin was placed in a 15 ml reactor and treated twice with 7 mL NMP and then filtered.
B2. Activated Amino Acids (3.0 eq) and 1-hydroxybenzotriazole (4.0 eq) were weighed and dissolved in NMP having 2.5 volumes thereof in a reactor and cooled to 0 ° C. DIC was then added (3.0 eq) and the mixture was stirred for 15 minutes.

B3. Coupling Next, the solution prepared in B2 was added to the reactor of B1. The reactor was washed once with 1 volume of DCM and added to the reactor, which was stirred at 25-30 ° C. for 1-3 hours. The completion of the reaction was determined by performing a Kaiser test on the samples. If the coupling reaction was not complete after 3 hours (Kaiser test positive), the reaction mixture was filtered and recoupled with a fresh solution of the activated amino acid. After completion of coupling, the reaction mixture was filtered and washed 4 times with NMP (5 volumes per wash).

B4. Removal of Fmoc-group
The resulting resin in B3 was filtered and then treated with 5 mL of a solution containing 25% by volume piperidine for 30 minutes. The resin was then washed 3 times with 5 mL NMP.

B5. Extension of the peptide chain After the introduction of each amino acid, steps B1 to B5 were repeated until the completion of the peptide chain.
The following Fmoc-amino acids were used for introduction of each individual amino acid: Fmoc-Ala-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Asn-OH, Fmoc-Asn (Trt) -OH, Fmoc-D -Cys (Trt) -OH, Fmoc-Cys (Trt) -OH, Fmoc-Gln-OH, Fmoc-Gln (Trt) -OH, Fmoc-Glu (tBu) -OH, Fmoc-Gly-OH, Fmoc-His (Trt) -OH, Fmoc-Hyp (tBu) -OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Met-OH, Fmoc-D-Phe-OH, Fmoc-Phe-OH, Fmoc-Pro -OH, Fmoc-Ser (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ser (Trt) -OH, Fmoc-D-Trp-OH, Fmoc-Trp -OH, Fmoc-D-Trp (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-Tyr (Clt) -OH, Fmoc-Val-OH, Boc-D -Cys (Trt) -OH, Boc-His (Trt) -OH, Boc-Lys (Boc) -OH, Boc-D-2-Nal-OH, Boc-D-Phe-OH, Boc-Ser (tBu) -OH.

C. General method for the acidic cleavage of peptides or protected peptide segments containing Fmoc- or Boc-groups at the N-terminus from CTC-resins , prepared as described above in B1-B5. The resin-bound peptide or peptide segment is washed 4 times with 5 mL NMP, 3 times with 5 ml IPA and finally 5 times with 7 ml DCM to remove any residual NMP or other basic components. Completely removed. The resin was then cooled to 0 ° C., filtered from DCM, and treated twice at 5 ° C. with 10 mL of 1-2% TFA / DCM solution. The mixture was then stirred at 0 ° C. for 20 minutes and filtered. The resin was then washed 3 times with 10 mL DCM.

  Next, pyridine was added to the filtrate (1.3 equivalents relative to TFA) to neutralize TFA. The cleavage solution in DCM is then mixed with the same volume of water. The resulting mixture is distilled under reduced pressure to remove DCM (350 torr at 28 ° C.). Peptides or peptide segments precipitate after removal of DCM. The resulting peptide is then washed with water and dried at 30-35 ° C. under a vacuum of 15 Torr.

Example 11. Synthesis of protected peptide bound to resin by condensation of N-terminal protected fragment with resin-bound C-terminal protected fragment
General procedure
To a solution of 0.15 mmol / ml N-terminal protected peptide fragment in DMSO / DCM (95: 5) is added 0.2 mmol HOBt and the resulting solution is cooled to 5 ° C. Next, 0.14 mmol of DIC is added and the mixture is stirred for 20 minutes at 15 ° C. and then added to 0.1 mmol of the resin-bound C-terminal fragment and stirred for an additional 6 hours at room temperature. The completion of the condensation reaction is checked by the Kaiser test. If the Kaiser test remained blue, a second condensation was performed to complete the condensation.

Example 12. Synthesis of a partially protected peptide by condensation in solution between a N-terminal protected fragment and a C-terminal protected fragment
General procedure
To a solution of 0.15 mmol / ml N-terminal protected fragment in DCM is added 0.2 mmol HOBt and the resulting solution is cooled to 5 ° C. Next, 0.15 mmol EDAC is added and the mixture is stirred at 15 ° C. for 20 minutes and then 0.15 mmol C-terminal protected fragment is added and an additional 2-5 at room temperature. Stir for hours. The completion of the condensation reaction is checked by HPLC. If incomplete condensation is observed, an additional 0.015 mmol of EDAC is added and the reaction is continued at room temperature for additional time.

Example 13 Simultaneous cleavage and deprotection of peptides from resin
General Method 1.00 g of protected resin-bound peptide prepared as described above was added with 20 mL of TFA / DTT / water (90: 5: 5) for 3 hours at 5 ° C and 1 at 15 ° C. Processing time. The resin is then washed 3 times with the cleavage solution and the combined filtrates are then concentrated in vacuo and the crude peptide is precipitated by addition of ether, washed several times with ether and constant over KOH. Vacuum dry to weight.

Example 14. Deprotection of peptides
General Method 1.00 g of the protected peptide prepared as described above was treated with 20 mL of TFA / DTT / water (90: 5: 5) for 3 hours at 5 ° C and 1 hour at 15 ° C. Processed. The resulting solution was concentrated in vacuo and then the deprotected peptide was precipitated by the addition of diisopropyl ether and washed three times with 10 mL diisopropyl ether. The resulting solid was vacuum dried (25 ° C., 15 Torr) to constant weight under KOH.

Example 15 Purification of Crude Peptide Isolation of Peptide
General Procedure The peptide solution obtained as above was concentrated in vacuo and ice water and ether were added. After separation of the organic phase, the remaining aqueous solution of the peptide is extracted twice more with ether and the resulting solution is blown with nitrogen or helium, filtered and semi-preparative column 10x25 cm, Lichrospher 100, RP-18, 12 microns. Added directly to (Merck); Phase A = 1% -TFA in acetonitrile, Phase B = 1% -TFA in water; or Kromasil. The HPLC fraction containing the purified peptide was concentrated in vacuo to remove as much contaminating acetonitrile as possible and lyophilized using a standard lyophilization program.

Examples 16-23 described below were performed using the procedure described above to prepare the compounds listed below.
Example 16. Lanreotide
Example 17 Insulin B-Chain
Example 18 Salmon calcitonin
Example 19. Octreotide
Example 20 Exenatide
Example 21. Pramlintide
Example 22. Tetracosactide (ACTH 1-24)
Example 23. Bivalirudin

Claims (10)

Formulas III-VI below:

Wherein Pr ¹ is H or an orthogonal amino protecting group for the resin and for other protecting groups;
Pr ² is H or an orthogonal hydroxyl protecting group for the resin;
R ³ and R ⁴ are each independently H or C _1-10 alkyl;
X, Y, Z and V are each independently an ortho, meta or para substituent and are selected from H, Cl, F, C _1-10 alkyl and C _1-10 alkoxy; and P is An insoluble support or insoluble linker-resin conjugate suitable for solid phase synthesis of peptides)
A resin joined body selected from: In the manufacturing method of the resin conjugate | zygote in any one of the formulas III-VI of Claim 1,
Preparing a hydroxyl-containing aminoalcohol that is not protected on at least one side chain of the aminoalcohol, or selectively deprotecting the protecting group of the hydroxylaminoalcohol on the side chain of the hydroxylaminoalcohol. And then attaching it to a suitable resin by reaction with a resin halide, where the resin is selected from trityl type resins and linkers and benzhydryl type resins; and alcohol or mask to mask unreacted resin halides Add thioalcohol;
A method comprising the steps.   A method for solid phase synthesis of biologically active free or partially protected peptides, cyclic peptides or peptaibols, wherein the resin conjugate according to claim 1 is functionalized in solid phase peptide synthesis. Using as a treated resin. Following formula:

(Where
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt and Mmt;
D- indicates the chirality of the amino acid according to the D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm and StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt and tBu; and the resin has the following formula:

Wherein X, Y, Z and V are each independently an ortho, meta or para substituent and are selected from H, Cl, F, C _1-10 alkyl or C _1-10 alkoxy And P is an insoluble support or insoluble linker-resin conjugate suitable for solid phase synthesis of peptides)
It is an acid-sensitive resin indicated by
A partially protected peptide represented by: Following formula:

(Where
A is H or an amino protecting group selected from Fmoc, Boc, Trt, Nps, Mtt and Mmt;
D- indicates the chirality of the amino acid according to the D-amino acid;
B is a thiol protecting group selected from Trt, Mmt, Acm and StBu;
C is H or Boc;
E is a hydroxy protecting group selected from Clt, Trt and tBu; and
The resin has the following formula:

Wherein X, Y, Z and V are each independently an ortho, meta or para substituent, and H, Cl, F, C _1-10 Alkyl or C _1-10 Selected from alkoxy; and
P is an insoluble support or insoluble linker-resin conjugate suitable for solid phase synthesis of peptides)
It is an acid-sensitive resin indicated by
A partially protected peptide represented by: 6. The peptide of claim 5 , wherein the peptide is octreotide bound to a resin. In the manufacturing method of octreotide,
Treating the peptide resin conjugate of claim 6 with a mild acid; and oxidizing the resulting peptide solution with a suitable oxidizing agent selected from air, hydrogen peroxide, DMSO or iodine;
A method comprising that. 8. The method of claim 7 , wherein the mild acid comprises a solution of trifluoroacetic acid optionally containing a scavenger. In the manufacturing method of octreotide,
Treating the peptide resin conjugate of claim 6 with a mild acid; and deprotecting, purifying, lyophilizing and obtaining octreotide with a purity greater than 99%;
A method comprising that. The method of claim 9 , wherein the mild acid comprises a solution of trifluoroacetic acid containing iodine.